The long term goal of our research is to understand the biochemical mechanism by which a eucaryotic gene activator stimulates transcription by RNA polymerase II (pol II). Knowledge of how an activator controls the level and diversity of gene expression would provide insights into the transcriptional events underlying development, disease and tumorigenesis. The current hypothesis posits that an activator stimulates transcription by interacting with targets in the general transcription machinery to promote assembly of a preinitiation complex at the beginning of a gene. The last step in preinitiation complex assembly is ATP-dependent melting of the startsite. The efficiency of preinitiation complex assembly is controlled by activator potency and synergy. Potency reflects the affinity of an activator for a target and synergy reflects the ability of two or more activators to contact multiple targets. This proposal addresses key aspects of the hypothesis using a model biochemical system, composed of GAL4-derived acidic activators, GAL4-responsive templates, and the HeLa cell general transcription machinery. Our Specific Aims are: 1. To determine how GAL4 derivatives assemble an active preinitiation complex. This problem will be addressed by forming complexes using purified transcription components and analyzing these complexes by DNA binding, immunoblotting and chemical crosslinking assays. 2. To identify and characterize the binding site of VP16 on its targets within the transcriptional machinery. Two new techniques will be used to analyze interaction of VP16 with its targets: chemical protease footprinting and phage display genetics. 3. To determine the role of synergy in controlling activator potency. The affinity of a multimerized VP16 activation domain for components of the general transcription machinery will be measured. 4. To determine the role of DNA melting in preinitiation complex assembly. A premelted startsite will be used as a substrate for in vitro transcription to determine if it can bypass the requirement for ATP, activator or certain pol Il accessory factors.